Matrix metalloproteinases (MMPs) are extracellular zinc- and calcium-dependent proteases, which are produced in a latent form and require activation for catalytic activity. Activation occurs at the cell surface and enables MMPs to degrade components of the extracellular matrix (ECM) at specific sites in the membrane surroundings. The most studied MMPs are the gelatinases, which include MMP-2 and MMP-9 that use gelatin, Type IV collagen and fibronectin as preferred substrates. While transcription of MMP-9 genes is transactivated by cytokines and growth factors, MMP-2 is constitutively expressed and unresponsive to phorbol ester and most cytokines. MMP-2 activation is regulated by MT1-MMP, which is a membrane-anchored MMP. MMP-9 activation is regulated by a protease cascade involving plasmin and stromelysin-1 (MMP-3).
Matrix metalloproteinases are responsible for much of the turnover of matrix components and as such are involved in normal as well as in pathological processes. MMPs have an important role in maintenance and remodeling of membranes and ECM, for example, in breaking down the extracellular matrix to allow cell growth and tissue remodeling during development and recovery from injury. They also play a role in processes such as ovulation, modulation of capillary permeability and in enabling cell migration to a site of inflammation.
MMPs are involved in many pathological conditions. For example, they are associated with pathogenic mechanisms in cancer such as invasion, metastasis and angiogenesis [Reviewed by Foda and Zucker (2001) Drug Discovery Today 6: 478-482]. MMPs play a part in progression of inflammatory conditions and diseases involving degradation of extracellular matrix, such as in stroke, hemorrhage, rheumatic diseases (e.g. arthritis), Crohn's disease, asthma, and in cerebrovascular and cardiovascular disorders [Mun-Bryce and Rosenberg (1998) J. Cerebral Blood Flow Metabolism 18:1163-72; Yong et al. (1998) TINS 21:75-80; Lukes et al. (1999) Mol. Neurobiol. 19:267-284]. Members of the MMP family have also been implicated in neurological diseases and conditions as being involved in demyelination and neuro-inflammatory processes. For example, MMPs have been associated with brain damage and ischemia, Guillain-Barré, multiple sclerosis, amyotrophic lateral sclerosis and Alzheimer's disease. The current notion is that inflammation leads to the production of cytokines, chemokines, growth factors and hormones that modulate MMPs production. Activation of MMPs and plasminogen activators (PAs) is an important regulatory step in the inflammatory response.
Members of another family of metalloproteinases, identified as “A Disintegrin And Metalloproteases” (ADAM), have, like members of the MMP family, multiple domains including a zinc-dependent catalytic domain and a N-terminus pro-domain that is responsible for maintaining the enzyme in an inactive state [Moss et al. (2001) Drug Discovery Today 6: 417-426]. It was shown that members of the ADAM family are involved in several different processing events including cleavage of substrates off the cell membrane surface (a phenomenon termed “shedding”). One member of the ADAM family is the TNFα-Converting Enzyme (TACE). TACE is found on the cell surface where it processes the membrane-bound TNFα, a pro-inflammatory cytokine, to its mature soluble form. Soluble TNFα, which is released in inflammatory conditions, can induce apoptosis. For example, TNF induces secretion and activation of MMP-9 in macrophages and glial cells and causes neuronal cell death in neuroinflammatory diseases and following brain injuries. TNF has also been shown to play a role in pathological conditions such as rheumatoid arthritis.
As potentially highly toxic proteolytic enzymes, the matrix metalloproteinases are tightly regulated at multiple stages, as follows:
i) Gene transcription—most MMPs are not constitutively expressed, but their transcription is controlled by various cytokines (e.g. IL-1, TNF) and growth factors (e.g. TGF-β, retinoic acid, FGF).
ii) Pro-enzyme activation—MMPs are normally produced in a latent form (pro-MMP) including a propeptide segment that generally must be removed to activate the enzyme.
iii) Inhibition of enzyme activity—There are at least four endogenous MMP-inhibitors known as tissue inhibitors of metalloproteinases (TIMPs), which bind to the enzyme and block its activity. Another known natural inhibitor of MMPs is the serum proteinase inhibitor α-macroglobulin.
Several synthetic inhibitors of MMPs have been described in various publications in the scientific and patent literature. Currently known inhibitors mainly include synthetic peptides and chelating agents [reviewed by Woessner J F Jr. in Ann N.Y. Acad. Sci. (1999) 30:388-403].
Some synthetic inhibitors of the MMP active site are peptidomimetics based on the sequence of peptides cleaved in collagen [Masui et al. (1977) Biochem Med. 17:215-21). Peptidic agents based on conserved peptide sequence derived from the pro-segment of human collagenase IV are disclosed by Stelter-Stevenson et al. [Am J Med Sci. (1991) 302:163-70] and in U.S. Pat. No. 5,270,447 to Liotta et al. Synthetic peptides isolated from phage display peptide libraries and cyclic peptides with MMP inhibitory activity are described by Koivunen et al. [Nat. Biothechnol (1999) 17: 768-74].
N-hydroxyformamide peptidomimetics useful as TACE and MMPs inhibitors are disclosed by Musso et al. [Bioorg Med Chem Lett (2001) 11: 2147-51].
Other polypeptides and peptoid compounds useful as metalloproteinase inhibitors are disclosed in U.S. Pat. Nos. 4,263,293 and 4,297,275 to Sundeen et al., in U.S. Pat. Nos. 4,371,465, 4,371,466 and 4,374,765 all issued to McGregor, and in U.S. Patent Publication No. 2002/0090654 to Langley et al.
Non-peptidic MMP-inhibitory compounds are disclosed in U.S. Pat. No. 4,950,755 to Witiak et al. and in U.S. Pat. No. 5,866,570 to Liang et al.
MMPs inhibitors comprising targeting moieties and chelators are disclosed in International Patent Publication No. WO 01/60820 of Dupont Pharmaceuticals Company, and in International Patent Publication No. WO 02/053173 to Kimberly-Clark Worldwide, Inc.
Matrix metalloproteinases as well as other members of the ADAM family are inhibited by chelating agents. Most of these chelating agents are natural and synthetic hydroxamate compounds and derivatives thereof such as succinyl hydroxamate, sulfonamide hydroxamate etc. [reviewed by Woessner, J. F. Jr. (1999) in Annals New York Academy of Sciences 30: 388-403]. For example, the synthetic hydroxamates batimastat (BB-94; Invest New Drugs (1996) 14:193-202) and its orally bioavailable analogue marimastat have been shown to inhibit spread and growth of malignant tumors in animals. These compounds are currently examined in advanced clinical trials.
Among the compounds that have been shown as MMPs inhibitors are also antibiotics such as tetracyclines and their chemically modified analogs (Golub et al. (1983) J Periodontal Res. 18:516-26; U.S. Pat. No. 4,704,383 to McNamara et al.; U.S. Pat. No. 5,837,696 to Golub et al.].
Most of the above-mentioned agents are non-specific inhibitors of metalloproteinases and other metal-ion dependent proteases.
Calpains are members of another family of proteases. These are cytosolic enzymes which are calcium-dependent cysteine proteases. Calpains predominantly exist within cells as inactive proenzymes and are converted into their active forms in the presence of elevated intracellular calcium levels. Upon binding of calcium, the precursor enzyme goes through a self-digestion process that results in release of the activated calpain.
A wide range of proteins serves as substrates for calpain including cytoskeletal, membrane and regulatory proteins. Calpain participates in a number of normal cellular signal transduction systems as well as in pathological conditions. For example, calpain activation has been associated with ischemia and neuronal cell death such as those caused by stroke and traumatic brain and spinal cord injuries [Bartus et al. (1995) Neurol. Research 17:249-258]. Calpain proteolytic activity has also been implicated in several neurodegeneration diseases and conditions, including Alzheimer's Disease, Parkinson's disease, Huntington's disease and Pick's disease.
Presently known natural and synthetic calpain inhibitors, including both peptidic and non-peptide molecules, are reviewed by Wang and Yuen [“Calpain inhibition: an overview of its therapeutic potential” in Trends Pharm. Sci. (1994) 15, 412-419] and by Donkor [“A survey of calpain inhibitors” in Curr. Med. Chem. (2000) 7:1171-88]. Known calpain inhibitors include polypeptides which mimic peptide sequences of the natural inhibitors calpastatin and kininogen [for review, see Wang and Yuen (1994) Trends Pharm. Sci. (1994) 15, 412-419].
Compounds which are sulfonamide derivatives and ketone derivatives that possess inhibitory activity against cysteine proteases are disclosed, respectively, in U.S. Pat. Nos. 5,506,243 and 5,639,783 both to Ando et al. Calpain inhibitors which are di-peptide alpha-keto esters, alpha-keto amides and alpha-keto acids are described by Li et al. [J. Med Chem (1993) 36: 3472-80]. Several other classes of calpain inhibitors are disclosed by Bartus et al. in International Patent Publication no. WO 92/11850.
Most commercially available calpain-inhibitors are compounds based on peptide structures that interact with the substrate-binding site of the enzyme. Many of these compounds are non-specific and inhibit a wide variety of proteases in addition to calpain. Moreover, most of the known inhibitors that were active in vitro, were found ineffective in inhibiting calpain in-vivo, in particular in the CNS, as being poor membrane permeants. Furthermore, almost all MMPs-inhibitors tested for treating pathological inflammatory conditions or cancers failed in in-vivo clinical studies.
Thus, there remains a long-felt need for effective, non-toxic agents which are specific inhibitors of critical proteases such as the MMPs and calpain.
BAPTA-Diesters
Stable lipophilic diesters of the divalent metal ion chelator 1,2-bis(2 aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) have been disclosed in the International Patent Publication No. WO 99/16741 of the same applicant. Also disclosed in this publication is the use of these compounds in pharmaceutical compositions useful for treating diseases and disorders related to excess of divalent metal ions. Among these diseases and disorders are ischemia, stroke, epilepsy and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.
At that time, however, the mechanism by which these chelating agents exert their neuroprotective effects has not been elucidated or disclosed. No indication or suggestion for the cellular targets affected by these chelators has been mentioned in the WO 99/16741 or any other publication.